Differentiation of eosinophilic granulocytes of carp (Cyprinus carpio L.).

Electron-microscopic studies were conducted to observe ultrastructural changes during differentiation of eosinophilic granulocytes in carp (Cyprinus carpio L.). Differentiation at the myelocyte stage was found to relate to specific granules made of dense and light fields. By maturation they assume a mosaic-like texture and in each granule of mature granulocytes, a light, central "internum" and a peripheral dense wrapper can be distinguished. The activity peroxidase and acid phosphatase is located in the internum and of peroxidase in the wrapper of the granules.     

Secretion in dissociated human pulmonary mast cells. Evidence for solubilization of granule contents before discharge

Mast cells were enzymatically dissociated from human lung fragments that had been sensitized with serum from human allergic to ragweed and were enriched by isopyknic and velocity gradient sedimentation. Electron microscope examination showed that the mast cells were well preserved at the end of the dissociation and isolation and that the majority of their secretory granules contained crystalline structures. These structures exhibited three patterns--scrolls, gratings, and lattices--which all could be found in the same granule. The period of crystalline structures was found to be bimodal, with maxima at 150 and 75 A. Both periods were observed in gratings that had been tilted and in scrolls that had been cut obliquely, indicating that the various gross patterns are composed of the same basic substructure. After the mast cells were stimulated by rabbit anti-human IgE to release histamine, the contents of the granule were transformed from a crystalline to an amorphous state, and only granules with amorphous contents were seen discharging from the cell. Clusters of intermediate filaments were present around the granules with amorphous contents, both deep in the cytoplasm and discharging at the cell surface. Discharge occurred both by fusion of granule membranes with the plasma membrane and by fusion of granule membranes with other granule membranes that ultimately were continuous with the plasma membrane. After discharge, the granule residue was fibrillar. Cells challenged with anti-human IgE in calcium-free medium neither released histamine nor demonstrated morphologic changes in their granules. We conclude that the crystalline state represents a storage form for materials that are solubilized before fusion of the granule membrane with the plasma membrane and discharge.     

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study.

The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 microns in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological and structural aspects of the giant starch granules from Phajus grandifolius

The morphology and structure of giant starch granules from the pseudo-bulbs of Phajus grandifolius were investigated, using a number of microscopy techniques together with synchrotron radiation microdiffration analysis. Most of the granules, which had sizes between 100 and 200 microm, occurred as ogival particles with the hilum or proximal end located at the apex of the granules. A small percentage of granules held a protuberance extending orthogonally to the underlying parent granule. Growth rings were observed in all granules: strongly curved close to the hilum, but planar toward the distal end of the granules or in the protuberances. Specific mechanical disruption followed by enzymatic digestion revealed the susceptibility of the disorganized parts of the growth rings, which were preferentially carved away during the digestion, leaving behind the better-organized domains. Microdiffraction analysis achieved with synchrotron radiation revealed the crystalline features of the granules and provided orientation maps of the amylopectin molecules in the various parts of the granules. In simple ogival granules the amylopectin molecules were uniformly oriented with their axes running from the hilum toward the distal end of the granule. In granules with a protuberance, the axes of the amylopectin molecules kept their direction in the parent granule, but took an orthogonal direction in the protuberance. The occurrence of these morphological and structural features is tentatively correlated with the mode of growth of these granules.     

Distribution of EPS and cell surface hydrophobicity in aerobic granules

This study described the distribution of extracellular polysaccharides (EPS) and hydrophobicity in aerobic granule as well as the essential role of EPS in maintaining the stable structure of aerobic granules. Aerobic granules showed a heterogeneous structure, which had an outer shell with high biomass density and an inner core having a relatively low biomass density. Results showed that the outer shell of aerobic granule was composed of poorly soluble and noneasily biodegradable EPS, whereas its core part was filled with readily soluble and biodegradable EPS. It was further found that the shell of aerobic granule exhibited a higher hydrophobicity than the core of granule. The insoluble EPS present in the granule shell would play a protective role with respect to the structure stability and integrity of aerobic granules.     

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.     

Membrane fusion during secretion: cortical granule exocytosis in sex urchin eggs as studied by quick-freezing and freeze-fracture

Exocytosis of cortical granules was observed in sea urchin eggs, either quick-frozen or chemically fixed after exposure to sperm. Fertilization produced a wave of exocytosis that began within 20 s and swept across the egg surface in the following 30 s. The front of this wave was marked by fusion of single granules at well-separated sites. Toward the rear of the wave, granule fusion became so abundant that the egg surface left with confluent patches of granule membrane. The resulting redundancy of the egg surface was accommodated by elaboration of characteristic branching microvilli, and by an intense burst of coated vesicle formation at approximately 2 min after insemination. Freeze-fracture replicas of eggs fixed with glutaraldehyde and soaked in glycerol before freezing displayed forms of granule membrane interaction with the plasma membrane which looked like what other investigators have considered to be intermediates in exocytosis. These were small disks of membrane contact or membrane fusion, which often occurred in multiple sites on one granule and also between adjacent granules. However, such membrane interactions were never found in eggs that were quick-frozen fixation, or in eggs fixed and frozen without exposure to glycerol. Glycerination of fixed material appeared to be the important variable; more concentrated glycerol produced a greater abundance of such "intermediates." Thus, these structures may be artifacts produced by dehydrating chemically fixed membranes, and may not be directly relevant to the mechanism by which membranes naturally fuse.     

Cortical granule-specific components are present within oocytes and accessory cells during sea urchin oogenesis

The coordinate expression of cortical granule-specific components in sea urchin oogenesis was studied using antibody probes. The components used to generate the organelle-specific antibodies included the whole cortical granule exudate, fertilization envelopes, hyalin, beta, 1-3,glucanase, and Ig8. Using immunolocalization techniques at both the light and electron microscopic levels, these molecules were found to be specific to cortical granules in three distinct cell types: developing oocytes, eggs, and accessory cells. In early oocytes, each of the cortical granule components are coordinately accumulated in the developing cortical granules dispersed throughout the cytoplasm. No other organelle within the developing oocytes or eggs contained detectable levels of any of these epitopes. In the somatic interstitial tissue of the ovary, cortical granule components also were accumulated specifically within cortical granule structures. Found only in select accessory cells, these cortical granules were indistinguishable in morphology and epitope composition from eggs and were contained within cytoplasmic aggregates termed mosaic globules. The mechanism of cortical granule concentration into mosaic globules is unknown, but this demonstration of common organelle constituents between oocytes and accessory cells may provide insight for such an understanding.     

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study

Summary The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 m in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled. From these findings, it is suggested that the granules of rat Clara cells consist of two types of granules of distinct origin; one appears to derive from condensing vacuoles of Golgi origin, whereas the other may be formed by membranefusions with small cytoplasmic vesicles of unknown source.     

Dynamic histology of the antral epithelium in the mouse stomach: II. Ultrastructure and renewal of isthmal cells

The isthmus of typical mucous units of the pyloric antrum was investigated in 3- to 4-month-old CD1 mice using light and electron microscopy as well as 3H-thymidine radioautography. On the average, the isthmus measured 25 microns in length and was composed of 36 isthmal cells and two enteroendocrine cells. Isthmal cells generally displayed features found in embryonic cells, such as many free ribosomes, scant organelles, and a large reticulated nucleolus, and were, therefore, at an immature stage of development. Isthmal cells could be devoid of secretory granules ("granule-free cells," 2%) or contain a few small, spherical, PA-Schiff-positive, mucous granules in their apex. The granules in some of the cells had a variegated appearance and a diameter averaging 235 nm ("mottled granule cells," 39%); in other cells, the granules had a large diameter, 278 nm, with a pale background and a dense core ("core granule cells," 28%); while in still others they were homogeneously dark and measured 264 nm ("dense granule cells," 12%). Finally, some cells included a mixture of core and dense granules ("mixed granule cells," 14%). One hour after a single injection of 3H-thymidine, 37% of the isthmal cells were labeled. Each of the five isthmal cell types could acquire label and, therefore, divide. After one or more days of continuous 3H-thymidine infusion, all isthmal cells were labeled. Their turnover time was estimated to be 16.1 hr (t1/2 = 11.2 hr). The isthmus is thus composed of several cell types which are turning over rapidly. While all are relatively immature, the various types are thought to represent different developmental stages in the life history of an isthmal cell. A model devised on this basis proposes that the granule-free cells are stem cells, from which mottled granule cells are derived. These in turn evolve into either the dense granule cells of the upper isthmus or the core granule cells of the lower isthmus, or into the mixed granule cells (which are believed to develop eventually into dense granule cells or core granule cells). Maintenance of a steady state requires that the rapid production of isthmal cells be associated with rapid emigration; the dense granule cells presumably going to the pit and the core granule cells to the gland. The turnover of isthmal cells is accordingly described as following a "bidirectional pattern" of renewal.     

Ca2+ influx does not trigger glucose-induced traffic of the insulin granules and alteration of their distribution

This study investigated mechanisms by which glucose increases readily releasable secretory granules via acting on preexocytotic steps, i.e., intracellular granule movement and granule access to the plasma membrane using a pancreatic beta-cell line, MIN6. Glucose-induced activation of the movement occurred at a substimulatory concentration with regard to insulin output. Glucose activation of the movement was inhibited by pretreatment with thapsigargin plus acetylcholine to suppress intracellular Ca2+ mobilization. Inhibitors of calmodulin and myosin light chain kinase also suppressed glucose activation of the movement. Simultaneous addition of glucose with Ca2+ channel blockers or the ATP-sensitive K+ channel opener diazoxide failed to suppress the traffic activation, and addition of these substances on top of glucose stimulation resulted in a further increase. Although stimulatory glucose had minimal changes in the intracellular granule distribution, inhibition of Ca2+ influx revealed increases by glucose of the granules in the cell periphery. In contrast, high K+ depolarization decreased the peripheral granules. Glucose-induced granule margination was abolished when the protein kinase C activity was downregulated. These findings indicate that preexocytotic control of insulin release is regulated by distinct mechanisms from Ca2+ influx, which triggers insulin exocytosis. The nature of the regulation by glucose may explain a part of potentiating effects of the hexose independent of the closure of the ATP-sensitive K+ channel.     

Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.     

Coexistence of renin and angiotensin II in epitheloid cell secretory granules of rat kidney

Summary The distribution of renin and angiotensin II (ANG II) in juxtaglomerular epitheloid cells of control and adrenalectomized rats was studied, using specific antisera and the protein A-gold technique in Lowicryl- and glycol methacrylate-embedded tissue.The matrix of virtually all mature secretory granules of epitheloid cells contains not only renin, but also ANG II. On adrenalectomy, the concentration of both renin and ANG II in the granule internum increases markedly, as indicated by the density of the immunolabel.Given the coexistence of renin and ANG II in the granule matrix, it is quite probable that, with each secretory event, a certain amount of ANG II is released together with renin. Further experiments will have to show if this amount of ANG II cosecreted with renin is sufficient to elicit immediate local intrarenal actions.ANG I, as well as angiotensinogen and converting enzyme, were not found in epitheloid cells. It is therefore inferred that ANG II is not generated intracellularly, but within the extracellular space and subsequently taken up by pinocytosis and incorporated into the secretory granules of epitheloid cells.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Ultrastructure of the eosinophil granule-internum; the problem of reversed relative density

Summary An electron microscope study was made of the specific granules of eosinophil leucocytes in mouse bone marrow. A crystal lattice was apparent in the granule-internum, with a repeating pattern of about 30 Å. The presence of this lattice in stained material in which the usual densities of internum and externum were reversed is taken as supporting the hypothesis that the reversal is a negative staining effect. Very occasionally a myelin-like inclusion was also seen in eosinophil granules.     

Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co- migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase- catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.     

Interaction of protein kinase C with chromaffin granule membranes: effects of Ca2+, phorbol esters and temperature reveal differences in the properties of the association and dissociation events

Interaction of protein kinase C with chromaffin granule membranes has been studied as a means of investigating the translocation of protein kinase C from cytosol to intracellular membrane surfaces, which is believed to occur during secretion. Protein kinase C in an adrenal medullary soluble fraction was found to bind reversibly to granule membranes in a Ca2+-dependent fashion. Association and dissociation events were sensitive to Ca2+ concentrations in the low micromolar range, and the Ca2+ sensitivity of both processes was increased when the membranes had been preincubated with the protein kinase C-activating phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (TPA). Binding of protein kinase C to granule membranes occurred at 0 and 37 degrees C, irrespective of whether the membranes had been preincubated with TPA. However, dissociation of protein kinase C from granule membranes that had been preincubated with TPA occurred only at 37 degrees C and not at 0 degree C, even though dissociation of the enzyme from membranes which had not been preincubated with TPA would occur at both 37 and 0 degrees C. These effects of TPA were not reproduced by 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD), a phorbol ester which does not activate protein kinase C. Soluble protein kinase C activity also associated with chromaffin granules in a Ca2+-dependent manner in an adrenal medullary homogenate, indicating that granules can compete with other intracellular membranes for the binding of protein kinase C. Results obtained with this model system differ from other systems where the interaction of protein kinase C with plasma membranes has been studied and have general implications for studies performed on the translocation of protein kinase C in intact cells and for the role of protein kinase C in stimulus-secretion coupling in the chromaffin cell.     

Characterization and evaluation of aerobic granules in sequencing batch reactor

In order to investigate the aerobic granules cultured under alternating aerobic and anoxic conditions, a sequencing batch reactor (SBR) was operated without the presence of a carrier material. Nitrification and denitrification occurred alternately in the SBR operation, with an increased nitrification efficiency of up to 97% and a high chemical oxygen demand (COD) removal efficiency of up to 95%. It was observed that physical characteristics of granule play an important role in the performance of the SBR process. Light microscopy was used to observe the time dependent development of the granules in the SBR. Based on the microscopic observations, some floc-like sludges remained in the form of a mixture with granules for 30 days of operation. Even though various granule sizes had been formed in the reactor after 50 days, the granule sizes were primarily from 1 +/- 0.35 to 1.3 +/- 0.45 mm, rarely exceeding 2 mm. The granules were analyzed by a combination of microelectrodes and fluorescent in situ hybridization (FISH), which provides more detailed information on what happens inside the granules. Based on their results, ammonia oxidizing bacteria (AOB) existed primarily in the upper and middle layers of the granule. Assuming a first-order reaction for nitrification, most of the nitrification is likely to occur from the surface to 300 microm into the granular thickness.     

Volutin Granules in Zoogloea ramigera

Zoogloea ramigera, a gram-negative bacterium found in activated sludge, formed volutin granules when excess orthophosphate was added to a phosphate-starved culture. These volutin granules were stainable by hydrogen sulfide after lead acetate treatment and extractable by N-perchloric acid but were not adsorbed by activated charcoal. They appeared to consist of inorganic polyphosphate. Optimum granule formation in the arginine broth required 10 g of glucose, 3 mg of phosphate, and 1 to 20 mg of magnesium per liter of medium. At an Mg(2+) concentration of 1 mg/liter, very large granules appeared which often appeared to fill the cell. An excess of glucose, orthophosphate, or magnesium reduced granule formation. In the absence of sulfate, moderate granulation occurred in arginine broth before the addition of excess orthophosphate; granulation did not increase after the addition of phosphate.     

The ultrastructure of the thyroid medullary carcinoma

The ultrastructure and the morphometrical pattern of secretory granules were studied in six cases of thyroid medullary carcinoma. The tumor cells were fusiform or polyhedral with irregular, mostly elongated nuclei. Phagolysosomes containing a crystalloid material, probably degraded lipoprotein complexes, degeneratively changed mitochondria, moderately developed rough endoplasmic reticulum and Golgi complexes were commonly found. Amyloid occurred as small fibrils in intercellular spaces. Marked dystrophic lesions of tumor cells surrounding amyloid fibrils were found. Numerous roundshaped electron-dense secretory granules were noticed in tumor cell cytoplasms. The morphometrical analysis showed that the size of granules oscillated between 60 and 450 nm with mean values ranging from 171.4 +/- 31.8 to 227.7 +/- 28.1 nm. Frequency distribution curves showed at least two peaks varying with the investigated case at different intervals. In two cases two distinct groups of granules were found within the same cells: one group of electron-dense, compact, smaller sized granules and another group of larger, finely granulated, less dense granules. In the other four cases the granule sizes were more homogeneous. These results might indicate that the granule size depends on the maturation degree and functional activity or that there are several kinds of granules specialized in the secretion of various substances.     

Quantitative analysis of the process and propagation of cortical granule breakdown in sea urchin eggs

Cortical granule breakdown in sea urchin eggs has been investigated with a video microscope system using Nomarski differential interference contrast optics, when induced by fertilization, microinjecting inositol 1,4,5-trisphosphate (IP3) or Ca-EGTA buffer solution into the egg, or perfusing a medium containing 1 mM Ca2+ to isolated cortices. The cortical granule increased up to 1.2 times in diameter and broke down within 40 msec. These values were almost constant among the three methods used to induce cortical granule breakdown. Upon fertilization, the cortical granule breakdown propagated over the egg surface at a speed of 3.3 microns/sec in Clypeaster japonicus eggs, which indicates that cortical granule breakdown propagated through the 3.3-microns-wide egg surface within 1 sec. In such a small area of the egg surface, however, it took much more than 1 sec for all cortical granules to break down because the maximal rate of breakdown was 7.6%/sec; that is, it took 9 sec and 18 sec for 50% and 90% respectively, of cortical granules to break down. Moreover, the rate did not simply decrease with time, and a shoulder was found during the reducing phase, which suggests that cortical granules are divided into fast and slow breakdown groups according to the responsiveness to the breakdown stimulus. The cortical granule breakdown induced by microinjecting the Ca-EGTA buffer and IP3 solutions propagated at 68 microns/sec and 35 microns/sec, respectively. The stimulus for cortical granule breakdown is discussed concerning the transient intracellular Ca2+ increase.